title: Galactonolactone oxidoreductase from Trypanosoma cruzi employs a FAD cofactor for the synthesis of vitamin C.
abstract: Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are interesting targets for drug therapy. The terminal step in ascorbate biosynthesis is catalyzed by flavin-dependent aldonolactone oxidoreductases belonging to the vanillyl-alcohol oxidase (VAO) protein family. Here we studied the properties of recombinant T. cruzi galactonolactone oxidoreductase (TcGAL), refolded from inclusion bodies using a reverse micelles system. The refolded enzyme shows native-like secondary structure and is active with both l-galactono-1,4-lactone and d-arabinono-1,4-lactone. At odd with an earlier claim, TcGAL employs a non-covalently bound FAD as redox-active cofactor. Moreover, it is shown for the first time that TcGAL can use molecular oxygen as electron acceptor. This is in line with the absence of a recently identified gatekeeper residue that prevents aldonolactone oxidoreductases from plants to act as oxidases.
authors.affiliation: Wagenigen University, Wagenigen, The Netherlands.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kudryashova
authors.forename: Nicole G H
authors.initials: NG
authors.lastname: Leferink
authors.forename: Ilse G M
authors.initials: IG
authors.lastname: Slot
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000096:biosynthesis*; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013400:Sugar Acids / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D013400:Sugar Acids; D005182:Flavin-Adenine Dinucleotide; C007813:galactonolactone; D010088:Oxidoreductases; D001205:Ascorbic Acid